Factor VIII is a coagulation factor acting as a co-factor in the generation of thombin, an essential component of coagulation. In the absence or insufficiency of functional factor VIII, individuals suffer from bleeding disorders, collectively called hemophilia A. There are two types of hemophilia A, depending from its origin, either genetic (spontaneous hemophilia) or acquired (acquired or autoimmune hemophilia). Spontaneous hemophilia A is a disease affecting males due the location of the factor VIII gene on the X chromosome. Women are carriers but do not suffer from bleeding disorders because of the presence of 2 X chromosomes. Spontaneous hemophilia is further divided into 3 subsets of patients, defined according to the level of circulating factor VIII: severe hemophilia (less than 1% factor VIII), mild hemophilia (1 to 5% factor VIII) and moderate (factor VIII concentrations between 5 and 10%).
Patients suffering from hemophilia require a substitution therapy by factor VIII. This is a continuing therapy for severe hemophilia A patients due to the increased risk of spontaneous, sometimes life-threatening bleeding, or intermittent in mild or moderate hemophilia patients in whom factor VIII is required when there is a trauma or surgery and an acute demand of increased factor VIII concentrations.
By far the main complication patients suffering from hemophilia A have to face is the emergence of antibodies towards the therapeutic agent (factor VIII) used to restore a functional coagulation.
There are currently 2 types of factor VIII used for replacement therapy, plasma-derived and recombinant. Plasma-derived factor VIII is produced from pools of human plasma and contains additional proteins and in particular the physiological chaperon of factor VIII, von Willebrand factor. Recombinant factor VIII is produced by genetic engineering and production by cells of animal or of human origin. Recombinant factor VIII is pure and do not contain von Willebrand factor. Vivid controversy is ongoing to decide whether there is a significant difference in the risk of eliciting an anti-factor VIII immune response when using either plasma-derived or recombinant factor VIII molecule. Whatever the situation, on average 25% of patients receiving factor VIII as a therapeutic agent raise antibodies which inhibit the activity of the replacement agent. Such antibodies are called factor VIII inhibitors.
There is no cure for factor VIII inhibitors. On empirical grounds, it has been demonstrated that the administration of very high doses of factor VIII on a daily basis can result in some cases in a disappearance of inhibitors. This therapy, called induction of immune tolerance, is not reliable in its success. The lack of surrogate markers able to predict the outcome of immune tolerance de facto limits its use in an attempt to eliminate the formation of factor VIII inhibitors. Moreover, the prohibitive cost related to tolerance induction is such that only a few patients can be considered for tolerance induction.
Factor VIII inhibitors are high-affinity specific antibodies, which implies the participation of T lymphocytes in their formation. The consequence of this is that the immune response is fully memorized, leaving populations of memory B cells, which upon stimulation, transform into plasmocytes producing antibodies, and memory T cells which maintain the capacity to mount further antibody response upon each subsequent exposure to factor VIII. Patients presenting with factor VIII inhibitors can not be treated with factor VIII not only because inhibitors neutralize factor VIII function and, likely, increase the clearance rate of factor VIII, but also because each further exposure to factor VIII increases the concentrations of such inhibitors.
Patent application WO 2009/101206 describes a method by which it is possible to eliminate the production of inhibitors by acting at the level of adaptive immunity, namely at the level of the interaction between factor VIII-specific T and B cells. This application describes how the risk of producing new inhibitors upon factor exposure can be eliminated, but also how existing inhibitors can be eradicated. However, we have unexpectedly discovered that factor VIII is a very potent activator of the innate immunity, which appears to be a pre-requisite for eliciting an adaptive response and inhibitors. Given that factor VIII has to be administered on a regular basis (e.g. 2 or 3 times a week for severe hemophilia A patients), and that therefore the risk of eliciting a new production of factor VIII inhibitors persists, there is an urgent need to define methods by which therapeutic factor VIII molecules could be produced, which have lost their capacity to activate the innate immunity.
The PCT application PCT/EP2011/070911 describes methods by which proteins with capacity to activate NKT cells can be transformed so as to loose such a capacity. Thus, NKT cells are part of the innate immune system, which is conventionally defined as lacking memorization. However, as described in PCT application PCT/EP2011/070911 NKT cells can recognize and be activated by the presentation of hydrophobic peptides by the CD1d molecule. As the peptide is derived from an antigen for which NKT cells are specific, this represent an antigen-specific innate immune system activation. The above-mentioned PCT application describes methods by which proteins showing the property to activate antigen-specific NKT cells can be modified by aminoacid substitution or deletion, thereby eliminating the capacity to bind to CD1d.
The present invention describes molecules of factor VIII obtained by the methodology described in PCT application PCT/EP2011/070911 which have lost their capacity to activate the innate immune system and, consequently, show a lack or significantly reduced capacity to active an adaptive immune response with production of inhibitors. The invention further describes the use of such factor VIII molecules for the treatment of patients in need for replacement therapy, and in particular severe hemophilia A patients. The present invention also discloses methods in which gene therapy using factor VIII molecules of the present invention can be used.